BRD2, BRD3, and BRD4 recruitment to GATA1-activated genes
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Mouse Gene 2.0ST arrays (Affymetrix) were hybridized per the manufacturer’s protocols. In microarrays, ERCC RNA Spike-In Mix (Ambion) was added to TRIzol-homogenized samples in proportion to cell number. Reverse transcriptase (RT)–qPCR was performed with iScript (Bio-Rad) and Power SYBR Green (Invitrogen). Quantitative polymerase chain reaction (qPCR) was run on ViiA7 System (Life Technologies) using Power SYBR Green (Invitrogen). We characterized the relationship of BETs with GATA1 on a genome-wide scale and demonstrate that BETs facilitate GATA1-mediated transcriptional activation but are largely dispensable for repression.
These inhibitors block the acetyl-lysine–binding pockets specifically of BET family bromodomains triggering their release from acetylated lysine residues on histones and transcription factors. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. BRD2 and BRD4 are essential for full GATA1 activity whereas BRD3 function overlaps with BRD2.
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BRD4 coordinates recruitment of pause release factor P-TEFb and the pausing complex NELF/DSIF to regulate transcription elongation of interferon-stimulated genes. Bromodomain and extra-terminal (BET) bromodomain inhibition activate transcription via transient release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein. Brd4 coactivates transcriptional activation of NF-kappaB via specific binding to acetylated RelA. Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting. Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes.
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- BET function is critical for GATA1-mediated gene activation, but not repression, by both facilitating GATA1 occupancy and subsequently activating transcription.
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What Gets Kept in a Cookie?
To further test the idea of functional overlap between BRD2 and BRD3, we examined erythroid maturation as reflected in hemoglobinization (red coloring) following GATA1 activation (Figure 6B). Similar to results in G1E cells, activation of erythroid gene expression was impaired by JQ1 whereas gene repression occurred normally (Figure 2E, supplemental Figure 7B). BET occupancy was not a strong predictor of JQ1 sensitivity overall, however, a weak relationship between JQ1 effects and BRD4 occupancy at promoters was observed (supplemental Figure 2B). Given the widespread expression and essential functions of BETs, it was initially surprising that BET inhibitors like JQ1 elicit cell- and gene-specific responses. Within the BET family, BRD2, BRD3, and BRD4 are ubiquitously expressed in mammalian tissues, whereas BRDT is testis-specific.
The mere presence of BETs at a given gene does not predict JQ1 response. In addition, other acetylated transcription factors might contribute to BET recruitment. BET-binding patterns are likely determined by association not only with acetylated GATA1 but also histone acetylation, which increases at many sites upon GATA1 activation. BRD3 is recruited to nearly all GATA1 sites whereas BRD4 occupies approximately one-half.
Role associated with BETs in GATA1 guests genome-wide
Here, the authors combine chemical proteomics with functional screens to assess the impact of oncogenic Src on the expressed kinome and identify SGK1 as a critical mediator of Src-induced cell transformation. Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions Beta II protein kinase C (βIIPKC) activation contributes to heart failure. Here, the authors develop a cell-free enzymatic prenylating system to generate isoprenyl pyrophosphate substrates directly from glucose and produce both common and rare cannabinoids at >1 g/L. A cell-free platform for the prenylation of natural products and application to cannabinoid production